Daniel:Notebook/PosSequencing/2017-6-26: Difference between revisions

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>Djacobse
(Created page with "=Beadless emPCR (Started Thursday 6/22/17)= Back to calendar Since the results on Daniel:Note...")
 
>Djacobse
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<ol type="A">
<ol type="A">
<li>Make the following master mixes</li>
<li>Make the following master mixes</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#B1A0C7;font-size:12pt;font-weight:bold" align="center"
| width="210" height="30" | Reagent
| width="95" | uL in Single Rxn
| width="95" | Master Mix P2 (3.2X)
| width="95" | Master Mix BioU (3.2X)
| width="95" | Master Mix Dual (5.2X)
|- style="font-size:12pt"
| height="15"  valign="bottom" | Primer 2 (10 uM)
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 3.2
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 5.2
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | BioU Primer (10 uM)
| align="center" align="center" valign="bottom" | 1
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 3.2
| align="center" align="center" valign="bottom" | 5.2
|- style="font-size:12pt"
| height="15"  valign="bottom" | 2X Kapa SYBR Master Mix
| align="center" align="center" valign="bottom" | 20
| align="center" align="center" valign="bottom" | 64
| align="center" align="center" valign="bottom" | 64
| align="center" align="center" valign="bottom" | 104
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | nfH2O
| align="center" align="center" valign="bottom" | 16
| align="center" align="center" valign="bottom" | 51.2
| align="center" align="center" valign="bottom" | 51.2
| align="center" align="center" valign="bottom" | 78
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total* (+ 5 uL sample=40)
|style="font-weight:bold" align="center" align="center" valign="bottom" | 35
|style="font-weight:bold" align="center" align="center" valign="bottom" | 118.4
|style="font-weight:bold" align="center" align="center" valign="bottom" | 118.4
|style="font-weight:bold" align="center" align="center" valign="bottom" | 192.4
|}


<li>Aliquot 35 uL master mix to appropriate lanes</li>
<li>Aliquot 35 uL master mix to appropriate lanes</li>

Revision as of 15:49, 26 June 2017

Beadless emPCR (Started Thursday 6/22/17)

Back to calendar

Since the results on Friday showed viable product (at least in the 100 nM sample), I'm going to finish up that experiment with strand denaturation and single primer qPCR.

Protocol

  1. Strand Separation (DNA Denaturation)
    1. Add 500 uL KOH buffer (pH 12.0)
    2. Spin down sample at 13,000rpm for 1 minute
    3. Remove all but 40uL buffer
    4. Repeat KOH steps A-C once more
    5. Add 500 uL 4X SSC buffer (pH 12.0)
    6. Spin down sample at 13,000rpm for 1 minute
    7. Remove all but 10uL buffer
    8. Resuspend in 80 uL 2X SSC buffer
  2. Single primer qPCR
    1. Make the following master mixes
    2. Reagent uL in Single Rxn Master Mix P2 (3.2X) Master Mix BioU (3.2X) Master Mix Dual (5.2X)
      Primer 2 (10 uM) 1 3.2 0 5.2
      BioU Primer (10 uM) 1 0 3.2 5.2
      2X Kapa SYBR Master Mix 20 64 64 104
      nfH2O 16 51.2 51.2 78
      Total* (+ 5 uL sample=40) 35 118.4 118.4 192.4
    3. Aliquot 35 uL master mix to appropriate lanes
    4. Add 2 uL sample according to plate layout below
    5. File:PlateLayout-Beadless-emPCR-singleprimer-20170626.png
    6. Run the following thermocycler program
      1. 95C 3 min
      2. 95C 3 sec
      3. 45C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x90
      7. 72C 2 min
      8. 16C hold